A. Field of the Invention
This invention relates to the production of biologically active amphiphilic peptides by recombinant techniques in bacterial host cells in which the peptide is expressed in high yield and is easily recovered. Despite the antimicrobial properties of the amphiphilic peptides, the claimed method avoids proteolytic degradation of the peptides and toxic effects on the host bacterial cells by expressing the peptides as fusion proteins in protease-deficient E. coli cells.
B. Description of the Prior Art
The development of recombinant DNA techniques and their application to the genetic engineering of microorganisms has dramatically changed our understanding of molecular and cellular biology during the past 20 years. These same techniques have also found useful applications in the production of rare or expensive biological molecules such as peptides and proteins of therapeutic value. Heterologous gene expression of therapeutic peptides and proteins has often been successful in organisms as diverse as bacteria, yeast, mammalian cells and, more recently, transgenic plants and animals.
The first choice for expressing therapeutic peptides and proteins has normally been the gram negative bacterium Escherichia coli. See, for example, Methods in Enzymology, Vol. 195 (D. Goeddel, ed.; Academic Press, New York, N.Y.; 1990); the description of the successful large scale production of recombinant human glucagon in E. coli by K. Yoshikawa et al. in Journal of Protein Chemistry 11, 517-525 (1992); and the expression of a recombinant salmon calcitonin precursor in E. coli by M. V. L. Ray et al. in Bio/Technology 11, 64-70 (1993).
There are, however, potential limitations in heterologous gene expression in E. coli, including toxicity of the foreign peptide or protein to the host cell, poor codon usage in the heterologous gene relative to the codon usage bias of the host cells, improper folding of recombinant gene products, and failed or inappropriate post-translational modification (e.g., glycosylation) of foreign proteins. These genetic engineering barriers have been studied and overcome to some degree. See, for example, D. V. Goeddel in Methods in Enzymology, Vol. 185 (D. V. Goeddel, ed., Academic Press, San Diego, Calif.; 1990), pp. 3-7 and A. L. Goldberg and S. A. Goff in Maximizing Gene Expression (W. Reznikoff and L. Gold, eds., Butterworth Publishers, Boston, Mass.; 1986), pp. 287-314.
Nonetheless, difficulties remain, particularly when expressing toxic peptides in E. coli. See, for example, the work of J. Lama and L. Carrasco on expression of toxic poliovirus polypeptides in E. coli in Journal of Biological Chemistry 267, 15932-15937 (1992) and Biochem. Biophys. Research Comm. 188, 972-981 (1992). These researchers found that expression of certain poliovirus proteins such as 3AB can be particularly toxic to E. coli hosts even when expressed in a highly regulated gene expression system such as the pET vectors developed by F. W. Studier and coworkers (cf. F. W. Studier et al., in Methods in Enzymology, Vol. 185 (D. V. Goeddel, ed., Academic Press, San Diego Calif. 1990), pp. 60-88).
Another limitation in heterologous gene expression in E. coli is the degradation of the proteins expressed. Many small peptides such as hormones and certain toxins are more susceptible to proteolytic degradation than large proteins. The proteolytic degradation of such peptides may be due to their relatively small size or to their lack of a highly ordered tertiary structure that would resist proteolytic degradation. See, for example, S. Gottesman in Methods in Enzymology, Vol. 185 (D. V. Goeddel, ed., Academic Press, San Diego, Calif.; 1990), pp. 119-129 and A. L. Goldberg and S. A. Goff in Maximizing Gene Expression (W. Reznikoff and L. Gold, eds., Butterworth, 1986), pp. 287-314.
Proteolytic degradation of expressed peptides may be reduced by producing peptides in the cytoplasm of bacteria as "inclusion bodies." Inclusion bodies are electron dense particles that consist of the recombinant protein and non-reducible polymers. See C. H. Schein, Bio/Technology 7, 1141-49 (1989). Unfortunately, however, useful peptides or proteins must be released from the inclusion bodies, requiring the use of strong chaotropic reagents, such as 6M urea or 8M guanidinium HCl. Such peptides and proteins may also require correct refolding or disulfide bond formation. Thus, the production of soluble recombinant peptides or proteins is preferred for the recombinant production of large quantities of therapeutically useful peptides or proteins, leaving unresolved the problem of proteolytic degradation of soluble peptides or proteins.
Another successful approach to reducing proteolytic degradation of expressed recombinant proteins or peptides is the use of protease-deficient host cell strains. It is widely understood in genetic engineering that protease-deficient host cell strains can increase yields of recombinant proteins, but there are no general means by which to predict what protease-deficient host cell strains are preferred in given instances. See, e.g., E. Bibi et al., Proc. Natl. Acad. Sci. (U.S.A.) 90, 9209-9213 (1993) on the utility of using E. coli ompT.sup.- host strains for expressing a recombinant multidrug resistance (mouse mdr1) gene, and D. M. Alexander et al., Protein Exp. and Purif. 3 204-211 (1992) on the utility of E. coli lon.sup.- host strains for expressing recombinant fibroblast growth factor. Bibi et al. found that ompT.sup.- hosts allowed stable expression of multidrug resistance genes in E. coli whereas two lon.sup.- host cell strains did not. By contrast, D. M. Alexander et al. obtained satisfactory expression of fibroblast growth factor in a lon.sup.- host cell strain. In both of these references, the site of recombinant protein appearance (E. coli outer membrane for the mouse mdr1 gene product and cytoplasm for fibroblast growth factor) is associated with the preferred protease deficiency. That is, the ompT protease, also known as protease VII, is found in the outer membrane and lon is the major cytoplasmic ATP-dependent protease of E. coli. See K. R. Rupprecht et al., J. Bacteriology 153, 1104-1106 (1983); G. Gordon et al., Mol. Gen. Genet. 193, 414-421 (1984); and A. L. Goldberg and S. A. Goff in Maximizing Gene Expression, loc. cit.
A further alternative successful approach to stabilizing foreign peptide gene products which are inherently unstable or toxic is to express them fused to a host cell protein or to a virally-encoded protein which displays stability in the relevant host cell. There is an extensive literature on protein fusions, especially in the gene expression host E. coli. See, e.g., Methods in Enzymology, Vol. 185 (op. cit.). Commonly used E. coli fusion protein partners include E. coli maltose binding protein (malE), anthranilate synthetase (trpE), .beta.-galactosidase (lacZ) and ribulokinase (araB), as well as Staph. aureus protein A, glutathione S-transferase of Schistosoma japonicum and the bacteriophage products of the cI gene of bacteriophage lambda and gene 10 of bacteriophage T7. See, for example, D. B. Smith et al., Proc. Natl. Acad. Sci. (U.S.A.) 83, 8703-8707 (1986); J. H. Nunberg, U.S. Pat. No. 4,701,416 (issued Oct. 20, 1987); C. di Guan et al., Gene 67, 21-30 (1988); J. C. Edman et al., U.S. Pat. No. 4,820,642 (issued Apr. 11, 1989); F. W. Studier et al. in Methods in Enzymology, Vol. 185 (op. cit.), pp. 60-88; and F. W. Studier et al., U.S. Pat. No. 4,952,496 (issued Aug. 28, 1990).
Even fusions with the, for example, male maltose binding protein, however, are not always stable. For example, despite fusing CD4 (the HIV receptor protein on human lymphocytes) to the periplasmic form of maltose binding protein (MBP) of E. coli, the N-terminal 177 amino acids of CD4 underwent significant degradation when expressed in E. coli. See Szmelcman et al., J. Acquired Immune Deficiency Syndromes 3, 859-872 (1990).
Despite the observation of instability in some recombinant fusion proteins, several groups have used recombinant processes to express potentially toxic antimicrobial peptides as fusion proteins. For example, J. Lai et al. in World Patent Application WO 86/04356 (published Jul. 31, 1986) and U.S. Pat. No. 5,028,530 disclose the recombinant synthesis of free acid forms of natural and mutant amphiphilic and antimicrobial cecropin peptides, isolated from the moth Hyalophora cecropia, as fusion proteins with the E. coli araB gene product in E. coli. These proteins were expressed as inclusion bodies. As set forth above, a significant disadvantage of inclusion bodies is the requirement for chaotropic agents to release the protein.
K. L. Piers et al., Gene 134, 7-13 (1993) disclose the use of E. coli recombinant expression systems to produce fusion proteins containing antimicrobial peptides and enumerate the advantages of such systems. However, the fusion proteins isolated by K. L. Piers et al. were expressed either as inclusion bodies in E. coli or as secreted proteins in Staph. aureus. As set forth above, a significant disadvantage of inclusion body formation in the teaching of J. Lai et al. or K. L. Piers et al. is the requirement for chaotropic agents to release the fusion protein. Further, Staph. aureus is not an acceptable host cell for producing recombinant therapeutic proteins because of its role as a significant human pathogen.
J. Lai et al. (loc. cit.) and K. L. Piers et al. (loc. cit.) do not disclose any benefit of producing cecropin or other antimicrobial peptide fusion proteins in soluble form, nor do they suggest any beneficial effects of expressing mutant or derivative forms of the natural cecropins or other antimicrobial peptides since, e.g., in the case of J. Lai et al., their mutant cecropin A peptides displayed the same antimicrobial profile as the natural cecropin A (see Table IV of WO 86/04356). These researchers also claim their fusion proteins are not bacteriocidal, do not disclose any potential benefits of using protease-deficient host cells, and offer no solutions to efficient recombinant production of fusion proteins which display bacteriocidal properties, especially soluble fusion proteins.
M. Hellers et al., Eur. J. Biochem 199, 435-439 (1991), report the expression and secretion of the natural amphiphilic and antimicrobial peptide cecropin A at high levels in recombinant baculovirus cultured on H. cecropia pupal cells. The secreted product was correctly processed and was amidated to a significant extent, but they experienced difficulty in expressing cecropin B in the same system. Moreover, this recombinant gene expression is poorly suited for large scale production of therapeutic peptides or proteins.
J. Jaynes et al. disclose in WO 88/00976 and WO 89/04371 the utility of expressing natural and derivative cecropin peptide sequences as well as other amphiphilic and antimicrobial peptides in transgenic plants such as tobacco and rice, but they do not disclose enabling technology for expressing amphiphilic and antimicrobial peptides in microbial cell hosts nor the utility of such microbial expression. Similar technology relating to expression of antimicrobial magainin peptides in plants is revealed in EP Application No. 0 472 987 by N. F. Bascomb et al. and in EP Application No. 0 552 559 by B. Scheffler and M. Bevan, but again neither enabling technology nor benefits of expression of magainin peptides in microbial hosts is disclosed.
K. L. Piers et al. (loc. cit.) disclose production of two antimicrobial peptides, human defensin NP-1 and a synthetic cecropin-melittin hybrid peptide designated CEME, as fusion proteins in E. coli, but noted significant instability of their fusion proteins unless they inserted additional DNA sequences encoding a defensin pre-pro sequence between the fusion protein and the antimicrobial peptide. They made use exclusively of the E. coli host strain DH5.alpha., which is protease proficient, and make no mention of any advantages conferred in using a protease-deficient E. coli host strain for expression of fusion proteins containing antimicrobial peptides. They also failed to obtain biologically active human defensin NP-1, which they ascribe to the inability of this defensin, which contains six cysteine residues, to form a proper disulfide array in their expression system. Thus, they did not identify any class of antimicrobial peptides with disulfide bridges which could be recovered in bioactive form in their expression systems without further biochemical processing or protein folding technology known in the art.
Thus, there is a need in the art for a process for the production of amphiphilic peptides by recombinant techniques in microbial hosts in which the peptide is expressed in high yield, may contain intramolecular disulfide bonds in the bioactive peptides, is not substantially degraded and is easily recovered, and the host cell is not otherwise negatively influenced to severely limit large scale production of such amphiphilic peptides.
There is also a need in the art for a process for the production of amphiphilic peptides which are modified post-translationally to improve their biological activity. It is known that amphiphilic peptides which are antimicrobial and/or inhibit the growth or proliferation of microbial pathogens have improved biological activity following amidation of their carboxyl terminus. See, e.g., J. H. Cuervo et al., Peptide Res. 1, 81-86 (1988) and J. Y. Lee et al., Proc. Natl. Acad. Sci. (USA) 86, 9159-9162 (1989). Enzymes such as peptidyl-glycine .alpha.-amidating monooxygenases are known which can amidate free carboxyl termini of peptides or proteins which terminate in a glycine residue, and such enzymes have been used to amidate the peptide hormone calcitonin, but the general utility of using amidating enzymes to post-translationally modify amphiphilic peptides is not known. Peptide substrate specificity in particular may be a strong limitation in modifying amphiphilic peptides. See P. P. Tamburini et al., Int. J. Peptide Protein. Res. 35, 153-156 (1990); Y. Iwasaki et al., Eur. J. Biochem, 201, 551-559 (1991); and M. V. L. Ray et al., Bio/Technology 11, 64-70 (1993). Similarly, chemical C-terminal amidation of unprotected peptides or proteins is generally difficult because of the potential for reaction of .epsilon.-amino groups on lysine or guanidino groups on arginine side chains with activated carboxyl terminal groups to form undesirable side products.